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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November 2012 to 18 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: off white powder
- Storage conditions: ambient, protected from light

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-9 weeks (males); 7-8 weeks (females) - treatment initiation
- Weight at study initiation: 296 - 356 g (males); 187 - 240 g (females) at treatment initiation.
- Housing: Animals were housed in polycarbonate cages with stainless steel grid tops and solid bottoms, with approximate dimensions of 61 x 43.5 x 24 cm. The animals were initially housed 2 or 3 per cage. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert. After mating, the males were re-housed with their original cage mates. Mated females were transferred to individual solid bottom cages. White paper tissue was supplied as nesting material from Day 20 of gestation.
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 22 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 air changes per hour (minimum)
- Photoperiod (hrs dark / hrs light): a 12 hour light/dark cycle was maintained
- Environmental enrichment: Chewing objects and hiding devices were provided to the animals as appropriate for psychological/environmental enrichment.

IN-LIFE DATES: From 26 November 2012 to 11 January 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) Carboxymethylcellulose (high viscosity) in Milli-Q water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
- The dosing formulations were prepared at weekly intervals, stored in a refrigerator set to maintain 4°C and dispensed daily. All formulations were used within the 8 day stability period that had been previously established. The dosing formulations were stirred for at least 30 minutes prior to and throughout dosing.
Any residual volumes were discarded following the completion of dosing each day.
On Day 38 of study, the refrigerator holding the test material was outside of the set range (2-8 °C) for approximately 12 hours, reaching a maximum temperature of 11 °C. As stability of the test material formulations has been established for up to 8 days at ambient temperature, this deviation was considered not to have impacted the integrity of the study.

- Dose volume: 10 mL per kg body weight.

- The volume administered to each animal was determined on each day by the weight of that animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each formulation, duplicate (0.1 mL) samples of top, middle and bottom samples were taken at each sampling time point and sent to the analytical laboratory for analysis (duplicate middle samples only obtained from Control formulations). Additional triplicate samples from the top, middle and bottom (middle sample only from control) were also taken as back-up samples. The results of the sample concentration analyses were considered acceptable if all results were within ± 10 % of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of ≤ 10 % for each group.

- Preparation of test solutions: approximately 0.1 mL of formulation was weighed into a digestion vessel and digested in approximately 20 mL of 69 % nitric acid. Once digested, the samples were transferred to a 100 mL volumetric flask with washings made to volume in 2 % nitric acid. The samples were further diluted, as necessary, in 2 % nitric acid to give sample solution concentrations within the range of the calibration standard used. Samples and standards were analysed by ICP-OES.

- Preparation of calibration standards: 20 mg of test material was added to a digestion vessel and digested in approximately 20 mL of 69 % nitric acid. Once digested the solution was transferred to a 100 mL volumetric flask with washings and made to volume in 2 % nitric acid to obtain a stock solution with a concentration of 0.200 mg/mL cerium trifluoride. The stock solution was diluted volumetrically in 2 % nitric acid to obtain target concentrations of approximately 0.00100, 0.00200, 0.00400, 0.00600, 0.00800 and 0.0100 mg/mL cerium trifluoride.

- ICP-OES conditions:
> Instrument: Perkin Elmer Optima 8000 ICP-OES
> Elements and wavelength: Ce (418.660 nm)
> No. of replicates: 3
> Delay time: 60 seconds
> Read time: set to auto
> Minimum read time: 1 second
> Maximum read time: 5 seconds
> Points per peak: 7
> Plasma conditions:
Plasma: 8 L/min
Auxiliary: 0.2 L/min
Nebuliser: 0.7 L/min
RF power: 1300 Watts
Plasma viewing mode: radial
> Peristatic pump sample flow rate: 1.50 mL/min
> Wash:
Frequency: between samples
Flow rate: 1.50 mL/min
Normal time: 30 seconds
Wash solvent: Milli-Q water
> Diluent: 2 % nitric acid
> Microwave digestion:
Ramp: 15 min to 180 °C
Hold: 15 min at 180 °C
Cool down: 30 min

- Calculations: a calibration curve was constructed by plotting the mean peak areas of the standards (including the calibration blank) against the relevant cerium trifluoride concentration in mg/mL. The concentration of cerium trifluoride in the test samples was determined by interpolation of this line.

Concentration of cerium fluoride in formulations (mg/mL) = (C x D x P) / W

Where
C = concentration of the sample solution obtained by interpolation from the calibration line (mg/mL)
D = dilution factor
P = density of the formulation (g/mL)
W = weight of aliquot (g)

- The limit of detection (LOD) estimated from an analyte response at the level of 3.3 times the ratio of the standard deviation of the blank to the slope of the calibration curve, was estimated to be 0.00000633 mg/mL cerium trifluoride.

- The limit of quantification (LOQ) estimated from an analyte response at the level of 10 times the ratio of the standard deviation of the blank to the slope of the calibration curve, was estimated to be 0.0000192 mg/mL cerium trifluoride.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Each female was transferred to the cage of its appropriate co-group male near the end of the work day, where it remained until mating had occurred or 14 nights had elapsed.
- Proof of pregnancy: Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrus observed in each lavage was recorded. The day of presence of sperm in such a lavage was designated Day 0 of gestation.
Duration of treatment / exposure:
The males were dosed daily for 4 weeks, starting 2 weeks prior to mating. The females were dosed daily 2 weeks prior to mating, throughout mating and through to at least Day 4 of lactation, with the following exceptions:
Animals 60 (100 mg/kg/day) and 78 (1000 mg/kg/day) were not dosed on Day 0 of lactation due to still being in the processing of littering. Dosing recommenced on Day 1 of lactation for these animals.
Animal 50 (0 mg/kg/day) was not dosed on Day 0 of lactation due to still being in the processing of littering; this animal was found dead later the same day.
Frequency of treatment:
Animals were dosed once daily.
Duration of test:
The males were killed when mating was completed and the animals had been dosed for at least 4 weeks.
The females and litters were killed between Day 5 and 7 of lactation.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected after a review of existing relevant toxicological data. Including a separate 14-day dose range finding study, where dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.
- Animal assignment: On arrival from the suppliers, the animals were housed in cages which were suspended on a series of racks. Male and female cages were racked separately. Cages were allocated to treatment group by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pretrial, all animals received a detailed clinical examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded one week prior to the start of treatment. From the start of treatment, the individual body weights were recorded daily.
* Animals 50 (0 mg/kg/day), 60 (100 mg/kg/day) and 78 (1000 mg/kg/day) were not weighed on Day 0 of lactation due to still being in the processing of littering.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating.
After mating, the female food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-4 of lactation.

GROSS PATHOLOGY: Yes
All adult animals surviving to scheduled euthanasia were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

ORGAN WEIGHTS: Yes
The following organs were weighed at necropsy for all adult animals surviving to terminal kill: Brain, Adrenal glands, Pituitary gland, Thyroid glands, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Thymus, Uterus.

HISTOPATHOLOGY: Yes
Representative samples of the following tissues were collected from all adult animals and preserved in 10 % neutral buffered formalin, unless otherwise indicated:
Aorta, Blood smear* (Animals sacrificed prematurely only), Bone marrow smear# , Bone marrow (femur, sternum), Femur, Rib, Sternum, Brain, Cervix, Eyes~, Adrenals, Harderian glands~ , Lacrimal glands, Mammary gland, Parathyroid glands, Pituitary gland, Salivary glands, Thyroids, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidneys, Large intestine (caecum, colon, rectum), Larynx, Liver, Lung, Mandibular lymph node, Mesenteric lymph node, Skeletal muscle, Nasal cavity, Optic nerve~, Sciatic nerve, Oesophagus, Ovaries, Oviducts, Pancreas, Pharynx, Skin, Small intestine (duodenum, ileum, jejunum), Spinal cord, Spleen, Stomach, Thymus, Tongue, Trachea, Ureter x2, Urinary bladder, Uterus, Vagina.
* Air dried
# Fixed in methanol and then air dried
~ Preserved in Davidson’s fixative

- Bone Marrow Smears
Duplicate bone marrow smears were taken at necropsy from all adult animals. Both bone marrow smears were stained using May-Grunwald-Giemsa as the Romanowsky stain. Bone marrow smears were not evaluated.

- Histopathology
The tissues, as listed above (except animal identification, nasal cavity, blood smears and bone marrow smears), were processed to paraffin wax block from selected animals. Sections were cut 4-6 µm thick, stained with haematoxylin and eosin from 5 females in the Control and High dose groups (the same animals that were used for laboratory investigations).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes, all per litter. Pups were examined for external visible abnormalities and for the presence of milk in the stomach.
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
All statistical tests were two-sided and performed at the 5 % significance level using in house software. Pairwise comparisons were only performed against the control group.
Selected body weight and food consumption data were analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.
Indices:
Fertility index, gestation index, birth index, live birth index and viability index were calculated.
Historical control data:
Historical control data was included for comparison. All observations in the test animals were within the historical background range.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
Animal 50 (0 mg/kg/day) was found dead on Day 0 of lactation. The animal had given birth to 16 live and 1 dead pups prior to being found dead, but had displayed no previous clinical signs. There were no necropsy findings in this animal. The reason(s) for death are not known, but as this was a Control animal it did not reflect an effect of treatment.
There were no clinical signs during the course of the study that were considered to be related to administration of test material.

BODY WEIGHT AND WEIGHT GAIN
Body weight gains were similar between Control animals and animals receiving test material.

FOOD CONSUMPTION
Food consumption was similar between Control animals and animals receiving test material.

MATING PERFORMANCE, FERTILITY AND DURATION OF GESTATION AND LITTER SIZE
Mating performance, fertility indices, corpora lutea and implantation counts, duration of gestation, and the mean number of pups born/live pups born per litter, were similar between Control females and females receiving test material.

OBSERVATIONS AMONG DAMS
All observations recorded for dams were common findings for this species and strain; therefore these observations were considered not to be related to treatment with test material.

ORGAN WEIGHTS
No test material-related organ weight changes were noted. There were isolate organ weight values that were different from Control. There were, however, no patterns, trends or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and unrelated to administration of test material.

GROSS PATHOLOGY
No test material-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat on this type of study, and/or were of similar incidence in Control and treated animals and, therefore, were considered unrelated to administration of test material.

HISTOPATHOLOGY
All microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in Control and treated animals and, therefore, were considered unrelated to administration of test material.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER SURVIVAL, LITTER AND PUP WEIGHTS
Total litter losses were noted in 1/10 females at 100 mg/kg/day, 1/10 females at 300 mg/kg/day and 2/10 at 1000 mg/kg/day versus 0/8 in Control females. The viability index was correspondingly lower than Control in these groups (88%, 86% and 78% respectively, versus 99% in Controls). Given that viability indices and percentage of litter losses in these groups were all within the historical background control range, these findings were considered to be incidental.
Litter weights and mean pups weights were similar between litters derived from Control females and litters derived from females receiving test material.

OBSERVATIONS AMONG PUPS
All observations recorded for pups were common findings for this species and strain; therefore these observations were considered not to be related to treatment with test material.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 2: Group Mean Litter and Pup Weight (g)

Day of lactation

Group / Dose level (mg/kg/day)

1 (0)

2 (100)

3 (300)

4 (1000)

LITTER

Day 1

83

81

91

85

Day 4

120

121

131

125

Mean of litter mean pup weight

MALES

Day 1

6.5

6.5

6.5

6.2

Day 4

9.4

9.9

9.5

9.1

FEMALES

Day 1

6.1

6.2

6.2

5.9

Day 4

9.0

9.4

9.3

8.6

Mean excludes litters where all pups died

 

Dose Formulation Analyses

Formulation analyses of formulations prepared for dosing during Week 1 of study were within the acceptance criteria at all concentrations.

The concentration of formulations prepared for dosing during Week 4 of study were above the acceptance criteria (± 10 % of theoretical concentration) at all concentrations; 14.0 %, 11.3 % and 19.0 % at 10, 30 and 100 mg/mL respectively. The Week 4 backup samples were analysed and all results were within the acceptance criteria. The reason for the initial (Week 4) results being outside of the acceptance criteria is not known.

 

Table 3: Formulation Analysis Results

Week 1

Dose group

Nominal conc (mg/mL)

Mean found (mg/mL)

% difference from nominal

1

0

0

na

2

10

10.1

1.0

3

30

30.6

2.0

4

100

108

8.0

Week 4

Dose group

Nominal conc (mg/mL)

Mean found (mg/mL)

% difference from nominal

1

0

0

na

2

10

11.4

14.0

3

30

33.4

11.3

4

100

119

19.0

Week 4 (back-up)

Dose group

Nominal conc (mg/mL)

Mean found (mg/mL)

% difference from nominal

2

10

9.66

-3.4

3

30

28.5

-5.0

4

100

101

1.0

na = not applicable

Applicant's summary and conclusion

Conclusions:
In females the maternal No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day due to a lack of any clear evidence of toxicity in the females up to the maximum concentration tested. No developmental effects were observed in pups from the high dose group, therefore the NOEL for developmental toxicity is considered to be 1000 mg/kg/day.
Executive summary:

The effects of the test material to maternal and developmental toxicity were investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 422. During the study groups of 10 male and 10 female rats were administered test material, by oral gavage, at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca 6 weeks of treatment).

In females, there were no signs of toxicity noted in any of the parameters or end points that were evaluated in this study. Furthermore, in all treated groups the birth index, live birth index and viability index was similar to Controls. In all treated groups the group mean litter weight and the mean of litter mean pup weight were similar to Controls throughout lactation. The type and distribution of observations amongst dams and their litters during lactation did not indicate any association with treatment. 

Therefore, the maternal No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day due to a lack of any clear evidence of toxicity in the females up to the maximum concentration tested. No developmental effects were observed in pups from the high dose group, therefore the NOEL for developmental toxicity is considered to be 1000 mg/kg/day.